Diagnostic method for screening complement regulatory protein levels to predict spontaneous abortion

ABSTRACT

The invention provides a method for the early detection of pregnancy failure, spontaneous abortion or premature birth by determinations of complement regulatory protein levels. A kit for use in rapid identification of these pregnancy complications is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of PCT/US01/14768, filed onMay 9, 2001, which claimed priority under 35 U.S.C. 119(e) of U.S.Provisional Application Serial No.: 60/204,344, filed May 15, 2000,which applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Twelve to twenty-two percent of all pregnancies end inspontaneous abortion, or miscarriage. Most data available suggestgenetic, hormonal or immunological factors associated with a majority ofspontaneous abortions. About 40% of all miscarriages are estimated to bethe result of chromosomal abnormalities. Various hypotheses have beenproposed for the remaining 60%, and a variety of tests and therapieshave been proposed for diagnosing and treating high-risk pregnancy. Forexample, high-risk pregnancies are evaluated using Doppler evaluation ofuterine artery blood flow (Caforio, L. et al., Fetal Diagn. Ther. (1999)14: 201-5), screening for and measurement of anti-paternal antibodies(Orgad, S., et al. Hum. Reprod. (1999) December 14: 2974-9), andmeasurement of MSAFP (maternal serum alpha-fetoprotein) levels, amongothers.

[0003] Despite the application of currently available technologies forscreening high-risk pregnancies, reliable methods have not been found.The pathology of spontaneous abortion is difficult to elucidate.Immunologically, a fetus is a semiallogenic graft and blunting of theimmune system is required to permit maintenance of the fetus by themother. Some have suggested that anti-paternal antibodies causerejection of the fetus by the maternal immune system, and one treatmentthat has been proposed for recurrent miscarriage is intravenousimmunoglobulin therapy (Daya, S., et al., Hum. Reprod. Update (1999) 5:475-82). Others have suggested that maternal blood flow to the placentacontributes to spontaneous abortion pathology.

[0004] One hypothesis that has been suggested for spontaneous abortionpathology is maternal rejection of the fetus due to complementregulatory proteins at the feto-maternal interface. Although one grouphas recently described a cell surface protein in mice that is directlyinvolved in fetal survival (Xu, et al., Science (2000) 287:498-501), nosuch molecule has been described in humans.

[0005] Differential expression of complement regulatory proteins at thefeto-maternal interface was investigated in 1992 by Holmes, et al.(Holmes, C. H., et al., Eur. J. Immunol. (1992) 22: 1579-85), whosuggested that differential expression might reflect the need forspecific functional activities within the placenta, and that theseproteins might be involved in pregnancy pathologies (Holmes, C. H., etal. (1992) Baillieres Clin. Obstet. Gynaecol. 6: 439-60). Fenichel etal. investigated complement regulatory proteins on human sperm,unfertilized oocytes, and pre-implantation embryos, concluding thatselective expression of complement regulatory proteins associated with alack of MHC class I antigens might represent an immune protectivemechanism for gametes and pre-implantation embryos during their travelthrough the female genital tract (Fenichel, P., et al., Contracept.Fertil. Sex (1995) 23: 576-80).

[0006] Pinpointing the pathologic mechanism and devising an accuratescreening technique for high-risk pregnancy has, however, beencomplicated by the complexity of the human immune and reproductivesystems. For example, Imrie et al. suggested that reduction in CD35(CR1) and CD55 (DAF) reflect increased levels of circulating immunecomplexes and consequent increased complement activation in pregnancy—anoutcome that would appear to put the fetus at risk in normal pregnancy,if complement activation is part of the pathology of spontaneousabortion (Imrie, H. J., et al. J. Reprod. Immunol. (1996) 31: 221-7).And, although the mouse model has provided valuable insights intomechanisms of immune response, there are fetomaternal tolerancemechanisms that are quite different between humans and mice, makingextrapolation from the mouse model to the human system problematic. Forexample, the Crry gene demonstrated by Xu et al. to determine fetalsurvival in mice is absent in humans.

[0007] While pathologic mechanisms associated with spontaneous abortionremain unclear, there remains a need for a simple and effectivescreening method for identifying high-risk pregnancies.

SUMMARY OF THE INVENTION

[0008] The present invention provides a method for diagnosing apredisposition for pregnancy failure, spontaneous abortion or prematurebirth in a pregnant patient by contacting a physiological fluidpotentially comprising a cell membrane-associated complement regulatoryprotein (CRP) from the patient with a anti-CRP antibody to form anCRP-antibody complex; and measuring the quantity of CRP-antibody complexin the physiological fluid as compared to a normal control level,wherein the quantity of CRP-antibody complex as compared to the normalcontrol is indicative for a predisposition for pregnancy failure,spontaneous abortion or premature birth. The CRP may be CD35 (complementreceptor type 1, CR1), CD46 (membrane cofactor protein, MCP), CD55 (alsodecay accelerating factor, DAF) or CD59 (membrane attack complexinhibitory factor, MACIF). The anti-CRP antibody may be immobilized on asolid surface. The anti-CRP antibody may be a detectable label or abinding site for a detectable label to form detectable complexes. Thedetectable label may be an enzyme label, or a fluorogenic compound. Thebinding site for the detectable label may be biotin, avidin orstreptavidin.

[0009] The present invention also provides a method for diagnosing apredisposition for pregnancy failure, spontaneous abortion or prematurebirth in a pregnant patient by contacting a physiological fluid from thepatient, wherein the fluid potentially comprises CRP, with a solidsurface having immobilized thereon anti-CRP antibodies, so that the CRPbinds to the anti-CRP antibodies; contacting labelled CRP, whichcomprises a detectable label or a binding site for a detectable label,with the solid surface, so that the labelled CRP binds to freeantibodies on the solid surface to form detectable complexes; anddetecting the complexes, wherein the quantity of the complexes isinversely proportional to the amount of CRP in the physiological fluid,wherein the quantity of CRP-antibody complex as compared to a normalcontrol is indicative for a predisposition for pregnancy failure,spontaneous abortion or premature birth. The CRP may be CD35, CD46, CD55or CD59. The detectable label may be an enzyme label or a fluorogeniccompound. The binding site for the detectable label may be biotin,avidin or streptavidin.

[0010] The present invention also provides an article of manufacture fordiagnosing a predisposition for pregnancy failure, spontaneous abortionor premature birth in a pregnant patient comprising packaging material,and a diagnostic kit and instructions within the packaging material,wherein the diagnostic kit comprises anti-CRP antibody, and a means formeasuring the quantity of CRP-antibody complexes in a physiologicalfluid from a patient, wherein the quantity of CRP-antibody complex ascompared to a normal control is indicative for a predisposition forpregnancy failure, spontaneous abortion or premature birth, and whereinthe instructions that indicate that the diagnostic kit can be used todiagnose a predisposition for pregnancy failure, spontaneous abortion orpremature birth in a pregnant patient. The CRP may be CD35, CD46, CD55or CD59. The kit may also contain a solid substrate. The anti-CRPantibody of the kit may be immobilized on a solid surface. The anti-CRPantibody may be a detectable label or a binding site for a detectablelabel to form detectable complexes. The detectable label may be anenzyme label. The detectable label may be a fluorogenic compound.Alternatively, the binding site for the detectable label may be biotin,avidin or streptavidin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1: Graph comparing endometrial DAF levels from women havingspontaneous or elective terminations of pregnancy. The range of DAFlevel for elective terminations were from 11-35, whereas the DAF levelsfor spontaneous abortions were from 0-13.9. Thus, the mean for electiveterminations was at 16.9, whereas the mean for spontaneous terminationswas at 3.6. These data give a statistically significant P value of lessthan 0.0001.

[0012]FIG. 2: Graph comparing DAF in spontaneous and elective abortions.The levels of DAF vary depending on whether pregnancy is viable ormiscarried. Levels below 10 are consistently found in spontaneouslyaborted pregnancies, which is predictive of which pregnancies willspontaneously abort.

DETAILED DESCRIPTION

[0013] The present invention provides a method for screening forhigh-risk pregnancies. The inventors have identified a cell-associatedcomplement-regulatory protein, the levels of which are correlated withthe occurrence of pregnancy failure, including spontaneous abortion andpremature birth. Premature birth is generally defined as deliverybetween the point of viability (about 22 weeks of gestation) and about33 weeks of gestation. Premature birth is associated with increasedperinatal mortality and morbidity. Early detection of premature birthcan delay labor long enough to permit effective preventive measures.Spontaneous abortion (miscarriage) is generally defined as anon-elective termination of pregnancy before the point of viability. Amethod is provided for quantifying levels of the complement-regulatoryprotein, and thereby identifying women who are at-risk for pregnancyfailure, spontaneous abortion or premature birth.

[0014] The present invention provides a method for determining thepresence or the level of CRP in a biological sample containing CRP. Forexample, decay accelerating factor (DAF, CD55) is a cell-associatedcomplement-regulatory protein that inhibits complement activation andthus protects the autologous tissues from the cytotoxic effects ofcomplement. DAF has been previously associated with paroxysmal nocturnalhemobloginuria (PNH), as decreased expression of DAF is correlated withpresence of the disease. However, PNH is characterized, in part, bylysis of red blood cells (RBCs), and it has been shown that RBCs whichdo not express DAF (Inab phenotype) often survive complement attack andlysis.

[0015] DAF is associated with the Cromer blood group antigens, which arelocated at various positions along the DAF molecule. It has beencharacterized as a glycosylphosphatidylinositol (GPI)-anchored membraneprotein that inhibits both the classical and alternative pathways ofcomplement activation, its chromosomal location has been identified asband q32.8,9 of human chromosome 1, and its sequence has been reported(Medof, M. E., et al. Proc. Natl. Acad. Sci. USA (1987) 84: 2007-11). Inconjunction with CD59 (protectin), CD46 (membrane cofactor protein), andCD35 (complement receptor type 1 (CR1)), it participates in theregulation of complement activity in the immune response.

[0016] The inventors have determined that DAF levels are useful forpredicting the risk of spontaneous abortion in pregnant women. Themethod for screening for high-risk pregnancy is described briefly asfollows. Urine, serum and/or saliva samples are collected from thepatient and are appropriately diluted, such as at a 1:10 to 1:100 level,in an appropriate buffer, such as PBS/BSA 1%, Tween 20 buffer. Serialdilutions of recombinant DAF can be used as a control standard.Microtiter plates are coated with an appropriate amount of anti-humanDAF antibody in an appropriate buffer, such as 2.5 μg/ml purifiedanti-human DAF antibody (CD55, Clone IA10, Pharmingen, San Diego,Calif.) in NaHCO₃ buffer, pH 8.2. After incubation, the plates arewashed and blocked, such as with 2% BSA in PBS. The samples are added tothe plates and incubated, such as for about 1.5 hours at roomtemperature. The plates were washed again, and biotynalated anti-humanDAF is added and incubated. After another washing step, streptavidinperoxidase was added and incubated. The reaction was developed by addingdiaminobenzidine (DAB). Plates were read in an ELISA reader andconcentration of unknowns were calibrated against the serial dilutionsof recombinant DAF standard. OD levels below about 14 are at risk for aspontaneous abortion. In particular, OD levels below about 10 areconsistently found in spontaneously aborted pregnancies.

[0017] Immunoassays

[0018] Examples of immunoassays that can be employed to determine therelative or absolute amount of CRP in a biological sample include thoseassay methods, formats and kits disclosed in U.S. Pat. No. 5,516,639.CRP analytes may be distinguished from other sample components byreacting the analyte with a specific receptor for that analyte. Assaysthat utilize specific receptors to distinguish and quantify analytes areoften called specific binding assays. The analyte of the presentinvention may be detected using a variety of specific binding assayformats. For example, various direct-binding assays may be employed. Insuch assays, receptors, such as antibodies or other binding proteins,are chemically coupled to make a cross-linked protein complex and thecomplex is immobilized on a solid phase. The immobilized chemicallycross-linked protein complexes are contacted with a sample containingthe analyte of interest, which may be distinguished from othercomponents found in the sample. For example, an antibody specific for aCRP can be immobilized on the surface of a solid substrate and used as acapture antibody to specifically bind to CRP in a biological fluid.Suitable substrates include particulate substrates such as polystyrenebeads, the wells of plastic microtiter plates, paper or synthetic fibertest strips and the like. The immobilized antibody can then be contactedwith the test sample to be assayed, e.g., with a biological fluid suchas plasma, serum, tears, urine or the like. The resulting antibody-CRPbinary complex can then be contacted with an anti-CRP antibody, such asrabbit anti-CRP serum.

[0019] Following binding of the analyte by the immobilized complex, thesolid phase may be washed and then contacted with an indicator, such asa labeled conjugate. The conjugate comprises an antibody, antibodyfragment, binding protein or analyte depending on assay format, and thelabel is a florescent, enzymatic, colorimetric, radiometric or otherlabeling molecule that is associated either directly or indirectly withthe conjugate. The label may be comprised of an enzymatic compound thatproduces florescence upon contact with a substrate. The extent to whichthe indicator is present on the solid support can be correlated with theamount of unknown analyte (see, for example, Tijssen, P., LaboratoryTechniques in Biochemistry and Molecular Biology, Practice and Theory ofEnzyme Immunoassay, pp. 173-219 (Chapter 10) and pp. 329-384 (Chapter14), Elsevier Science Publishers, Amsterdam, The Netherlands, (1985)).

[0020] An anti-CRP monoclonal antibody can be itself coupled to adetectable label of a binding site for a detectable label. For example,the antibodies can be labeled radioisotopically, e.g., by ¹²⁵ I, orconjugated directly to a detector enzyme, e.g., alkaline phosphatase orhorse radish peroxidase, or can be labeled indirectly with a bindingsite for a detectable label, e.g., via biotinylation. The biotinylatedantibody can then be detected by its ability to bind to a anavidin-linked enzyme. If the second antibody is biotinylated, a detectorenzyme conjugated to avidin will be subsequently added. The final stepfor detecting enzymes conjugated to monoclonal antibody or to avidin itsthe addition of a substrate appropriate for the enzyme to allowquantitative colorimetric detection of reaction product. The value (readin optical density units) can be converted to fmol of CRP by referenceto a standard curve generated in a control assay in which a standardextract of detergent-solubilized CRP is added in graded concentrationsto the immobilized anti-CRP monoclonal antibody.

[0021] The present invention may use many other assay formats, such ascompetitive immunoassays, bead agglomeration assays and sandwich-typeimmunoassays, such as ELISA, as would be recognized by the art.

[0022] In competitive assay formats, the solid phase containingimmobilized chemically cross-linked protein complexes with specificityfor a selected analyte is contacted with a sample presumably containingsuch analyte and with a specific competitive reagent. The specificcompetitive reagent may be a labeled analog of the analyte. In thisspecific embodiment, the labeled analog competes with the sample analytefor binding to a receptor immobilized on the solid phase.

[0023] In the alternative, an analyte may be coupled to a solid phaseand contacted with a sample and with a specific competitive cross-linkedprotein reagent, for example, a labeled receptor for the analyte. Inthis format, sample analyte competes with solid phase analyte forbinding with soluble labeled cross-linked receptor. In both embodiments,the amount of label bound to the solid phase after washing provides anindication of the levels of analyte in the sample. That is, the amountof analyte in a sample is inversely proportional to the amount ofanalyte in the sample.

[0024] Another embodiment of the present invention is a diagnostic kitfor detecting or determining the presence of CRP in a biological sample.Immobilized antibodies and labeled antibodies are conveniently packagedin kit form, wherein two or more of the various immunoreagents will beseparately packaged in preselected amounts, within the outer packagingof the kit, which may be a box, envelope, or the like. The packagingalso preferably comprises instruction means, such as a printed insert, alabel, a tag, a cassette tape and the like, instructing the user in thepractice of the assay format.

[0025] For example, one such diagnostic kit for detecting or determiningthe presence of CRP comprises packaging containing, separately packaged:(a) a solid surface, such as a fibrous test strip, a multi-wellmicroliter plate, a test tube, or beads, having bound thereto antibodiesto CRP; and (b) a known amount of antibodies specific to CRP, whereinsaid antibodies comprise a detectable label, or a binding site for adetectable label.

[0026] In one embodiment of the invention, a clinical test kit issupplied for use in a hospital or clinic. Such a kit consists, forexample, of a microtiter plate that is coated with an appropriate amountof anti-human DAF antibody and purified anti-human DAF antibody.Appropriate buffers may also be provided. Further, streptavidinperoxidase and diaminobenzidine (DAB) may be provided. When provided toa qualified technician or other health care worker in a hospital orclinic, the kit provides a method for assaying DAF levels from a samplesupplied by a patient who may or not be at risk for spontaneous abortionor miscarriage. By analyzing the levels of DAF in the sample, it ispossible to identify increased risk of miscarriage and to provideappropriate treatment and counseling.

[0027] Solid Supports

[0028] A solid support useful in the present invention is a matrix ofmaterial in a substantially fixed arrangement. Exemplary solid supportsinclude glasses, plastics, polymers, metals, metalloids, ceramics,organics, etc. Solid supports can be flat or planar, or can havesubstantially different conformations. For example, the substrate canexist as particles, beads, strands, precipitates, gels, sheets, tubing,spheres, containers, capillaries, pads, slices, films, plates, slides,etc. Magnetic beads or particles, such as magnetic latex beads and ironoxide particles, are examples of solid substrates that can be used inthe methods of the invention. Magnetic particles are described in, forexample, U.S. Pat. No. 4,672,040, and are commercially available from,for example, PerSeptive Biosystems, Inc. (Framingham Mass.), CibaCorning (Medfield Mass.), Bangs Laboratories (Carmel Ind.), andBioQuest, Inc. (Atkinson N.H.).

[0029] Indicator Labels

[0030] The labels used in the assays of invention can be primary labels(where the label comprises an element which is detected directly) orsecondary labels (where the detected label binds to a primary label,e.g., as is common in immunological labeling). An introduction tolabels, labeling procedures and detection of labels is found in Polakand Van Noorden (1997) Introduction to Immunocytochemistry, secondedition, Springer Verlag, N.Y. and in Haugland (1996) Handbook ofFluorescent Probes and Research Chemicals, a combined handbook andcatalogue Published by Molecular Probes, Inc., Eugene, Oreg. Primary andsecondary labels can include undetected elements as well as detectedelements. Useful primary and secondary labels in the present inventioncan include spectral labels such as fluorescent dyes (e.g., fluoresceinand derivatives such as fluorescein isothiocyanate (FITC) and OregonGreen™, rhodamine and derivatives (e.g., Texas red, tetramethylrhodamineisothiocyanate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA,CyDyes™, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P,³³P), enzymes (e.g., horse-radish peroxidase, alkaline phosphatase)spectral colorimetric labels such as colloidal gold or colored glass orplastic (e.g. polystyrene, polypropylene, latex) beads. The label may becoupled directly or indirectly to a component of the detection assay(e.g., the labeling nucleic acid) according to methods well known in theart. As indicated above, a wide variety of labels may be used, with thechoice of label depending on sensitivity required, ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions. In general, a detector which monitors ananalyte-receptor complex is adapted to the particular label which isused. Typical detectors include spectrophotometers, phototubes andphotodiodes, microscopes, scintillation counters, cameras, film and thelike, as well as combinations thereof. Examples of suitable detectorsare widely available from a variety of commercial sources known topersons of skill. Commonly, an optical image of a substrate comprisingbound analyte is digitized for subsequent computer analysis.

[0031] Preferred labels include those which utilize 1) chemiluminescence(using Horseradish Peroxidase and/or Alkaline Phosphatase withsubstrates that produce photons as breakdown products) with kits beingavailable, e.g., from Molecular Probes, Amersham, Boehringer-Mannheim,and Life Technologies/Gibco BRL; 2) color production (using bothHorseradish Peroxidase and/or Alkaline Phosphatase with substrates thatproduce a colored precipitate) (kits available from LifeTechnologies/Gibco BRL, and Boehringer-Mannheim); 3) hemifluorescenceusing, e.g., Alkaline Phosphatase and the substrate AttoPhos (Amersham)or other substrates that produce fluorescent products, 4) Fluorescence(e.g., using Cy-5 (Amersham), fluorescein, and other fluorescent tags);5) radioactivity using kinase enzymes or other approaches. Other methodsfor labeling and detection will be readily apparent to one skilled inthe art.

[0032] Fluorescent labels are highly preferred labels, having theadvantage of requiring fewer precautions in handling, and beingamendable to high-throughput visualization techniques (optical analysisincluding digitization of the image for analysis in an integrated systemcomprising a computer). Preferred labels are typically characterized byone or more of the following: high sensitivity, high stability, lowbackground, low environmental sensitivity and high specificity inlabeling. Fluorescent moieties, which are incorporated into the labelsof the invention, are generally are known, including Texas red,dixogenin, biotin, 1- and 2-aminonaphthalene, p,p′-diaminostilbenes,pyrenes, quaternary phenanthridine salts, 9-aminoacridines,p,p′-diaminobenzophenone imines, anthracenes, oxacarbocyanine,merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolylbenzene, 1,2-benzophenazin, retinol, bis-3-aminopyridinium salts,hellebrigenin, tetracycline, sterophenol, benzimidazolylphenylamine,2-oxo-3-chromen, indole, xanthen, 7-hydroxycoumarin, phenoxazine,calicylate, strophanthidin, porphyrins, triarylmethanes, flavin and manyothers. Many fluorescent tags are commercially available from the SIGMAChemical Company (Saint Louis, Mo.), Molecular Probes, R&D systems(Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.),CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp.,Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCOBRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka ChemicaBiochemikaAnalytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems(Foster City, Calif.), as well as many other commercial sources known toone of skill.

[0033] Most typically, the analyte is measured by quantifying the amountof label fixed to the solid support by the capture of the linked complexbetween analyte and receptor. Typically, the presence in the reactionmixture of an analyte-receptor complex will increase or decrease theamount of label fixed to the solid support relative to a controlreaction which does not comprise the analyte. Means of detecting andquantifying labels are well known to those of skill in the art. Thus,for example, where the label is a radioactive label, means for detectioninclude a scintillation counter or photographic film as inautoradiography. Where the label is optically detectable, typicaldetectors include microscopes, cameras, phototubes and photodiodes andmany other detection systems which are widely available.

[0034] Biological Samples

[0035] Biological samples that can be used in the present inventioninclude physiological fluids. Physiological fluids from patients includeplasma, serum, tears, urine, and the like.

[0036] The following examples are intended to illustrate but not limitthe invention.

EXAMPLES Example 1 Estimation of DAF in Biological Fluids by ELISA

[0037] Levels of DAF in the urine, serum and saliva obtained frompatients was determined by Enzyme linked immunoabsorbent assay (ELISA).Flat bottomed, 96-well microtiter plates (Immunolon I, Dynatech Labs,Chantilly, Va.) were coated with 2.5 μg/ml purified anti-human DAFantibody (CD55, Clone IA10, Pharmingen, San Diego, Calif.) in NaHCO₃buffer, pH 8.2. The plates were washed three times after overnightincubation at 4° C. and blocked for 2 hours at 37° C. with 2% BSA inPBS. Serial dilutions of recombinant DAF were used as standard. Urineand saliva samples were diluted at 1:10, and serum samples at 1:100 inPBS/BSA 1%, Tween 20 buffer and were incubated in plates for 1.5 hoursat room temperature. Plates were washed again three times as above.Biotynalated anti-human DAF (CD55, Clone IA10, Pharmingen, San Diego,Calif.) diluted in PBS was added and incubated for another hour. Afteranother washing step as above, streptavidin peroxidase was added andincubated for 30 minutes. The reaction was developed by addingdiaminobenzidine (DAB) (Sigma, St. Louis, Mo.). Plates were read in anELISA reader and concentration of unknowns were calibrated against theserial dilutions of recombinant DAF standard.

Example 2 Determination of Levels of Endometrial DAF

[0038] Endometrial samples were obtained from 51 pregnant women,including 34 undergoing elective and 17 undergoing spontaneousabortions. Six micrometer sections were stained with anti-human DAF IgGby immunohistochemistry. DAF expression was quantitated by acomputer-based image analysis system. The relative increase in the ODvalue was calculated as percent of increase in cumulative OD values. Thebaseline was measured in a parallel section where buffer replaced theantibody.

[0039] The relative increase in integrated optical-density of DAF in theendometrial tissue of patients undergoing spontaneous abortions was 2.7%(n=17). The density of DAF expression was substantially increased to16.75% (p<0.0001) in the endometrium of patients undergoing electivetermination of pregnancy (n=34). About 97% of pregnancies withendometrial DAF level lower than 10% terminated spontaneously.

[0040] Thus, endometrial DAF levels were significantly lower in thespontaneous abortion group suggesting that DAF protects thesemiallogenic conceptus from autologous complement cascade and lowlevels of DAF is responsible for spontaneous abortions in a significantnumber of women.

[0041] All publications, patents and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the scope of the invention.

What is claimed is:
 1. A method for diagnosing a predisposition forpregnancy failure, spontaneous abortion or premature birth in a pregnantpatient comprising: (a) contacting a physiological fluid potentiallycomprising a cell membrane-associated complement regulatory protein(CRP) from the patient with a anti-CRP antibody to form an CRP-antibodycomplex; and (b) measuring the quantity of CRP-antibody complex in thephysiological fluid as compared to a normal control level, wherein thequantity of CRP-antibody complex as compared to the normal control isindicative for a predisposition for pregnancy failure, spontaneousabortion or premature birth.
 2. The method of claim 1, wherein the CRPis CD35, CD46, CD55 or CD59.
 3. The method of claim 1, wherein theanti-CRP antibody is immobilized on a solid surface.
 4. The method ofclaim 1, wherein the anti-CRP antibody comprises a detectable label or abinding site for a detectable label to form detectable complexes.
 5. Themethod of claim 4 wherein the detectable label is an enzyme label. 6.The method of claim 5 wherein the detectable label is a fluorogeniccompound.
 7. The method of claim 4 wherein the binding site for thedetectable label is biotin, avidin or streptavidin.
 8. The method ofclaim 4 wherein the quantity of CRP-antibody has an OD level below 14.9. The method of claim 8 wherein the OD level is below
 10. 10. A methodfor diagnosing a predisposition for pregnancy failure, spontaneousabortion or premature birth in a pregnant patient comprising: (a)contacting a physiological fluid from the patient, wherein the fluidpotentially comprises a CRP from the patient with a solid surface havingimmobilized thereon anti-CRP antibodies, so that CRP present in thefluid binds to the anti-CRP antibodies; (b) contacting labelled CRP,which comprises a detectable label or a binding site for a detectablelabel, with the solid surface, so that the labelled DAF binds to freeantibodies on the solid surface to form detectable complexes; and (c)detecting the complexes, wherein the quantity of the complexes isinversely proportional to the amount of CRP in the physiological fluid,and wherein the quantity of CRP-antibody complex as compared to a normalcontrol is indicative for a predisposition for pregnancy failure,spontaneous abortion or premature birth.
 11. The method of claim 10,wherein the CRP is CD35, CD46, CD55 or CD59.
 12. The method of claim 10wherein the detectable label is an enzyme label.
 13. The method of claim10 wherein the detectable label is a fluorogenic compound.
 14. Themethod of claim 10 wherein the binding site for the detectable label isbiotin, avidin or streptavidin.
 15. An article of manufacture fordiagnosing a predisposition for pregnancy failure, spontaneous abortionor premature birth in a pregnant patient comprising packaging material,and a diagnostic kit and instructions within the packaging material,wherein the diagnostic kit comprises anti-CRP antibody, and a means formeasuring the quantity of CRP-antibody complexes in a physiologicalfluid from a patient wherein the quantity of CRP-antibody complex ascompared to a normal control is indicative for a predisposition forpregnancy failure, spontaneous abortion or premature birth, and whereinthe instructions that indicate that the diagnostic kit can be used todiagnose a predisposition for pregnancy failure, spontaneous abortion orpremature birth in a pregnant patient.
 16. The article of manufacture ofclaim 15, further comprising a solid substrate.
 17. The article ofmanufacture of claim 15, wherein the CRP is CD35, CD46, CD55 or CD59.18. The article of manufacture of claim 15, wherein the anti-CRPantibody is immobilized on a solid surface.
 19. The article ofmanufacture of claim 15, wherein the anti-CRP antibody comprises adetectable label or a binding site for a detectable label to formdetectable complexes.
 20. The article of manufacture of claim 19,wherein the detectable label is an enzyme label.
 21. The article ofmanufacture of claim 20, wherein the detectable label is a fluorogeniccompound.
 22. The article of manufacture of claim 21, wherein thebinding site for the detectable label is biotin, avidin or streptavidin.